Project summary The long term goal of our research is to understand and manipulate immune surveillance pathways. The antigen processing pathway yields thousands of peptide/MHC complexes (pMHC I) on the cell surface as potential ligands for CD8+ T cells to allow detection of viruses or cancer. It is now clear that generation of the normal pMHC I repertoire requires peptide trimming in the endoplasmic reticulum (ER) by ERAAP, the ER aminopeptidase associated with antigen processing. The ERAAP is targeted for immune evasion by viruses and polymorphisms in this gene are linked to many autoimmune diseases. It is therefore essential to monitor ERAAP activity to ensure effective immune surveillance by CD8+ T cells. We had discovered earlier that the unique FL9 nona peptide ? derived from the Fam49a/b proteins of unknown function ? was presented by the non-classical MHC Ib molecule called Qa-1b only in ERAAP- deficient cells. This FL9 peptide/Qa-1 complex, called QFL was recognized by QFL-specific CD8+ T cells which lysed target cells expressing this ligand. Thus QFL- T cells are a mechanism for detecting and eliminating cells with ERAAP-deficiency. The QFL-T cells are relatively abundant, and share key characteristics with other innate-like iNKT (invariant NK T) and MAIT (mucosal - associated invariant T) cells which are also restricted by non-classical MHC Ib molecules and respond to various microbial and self-antigens. Here we propose to fill gaps in our understanding of how the immune system monitors the fidelity of antigen processing in the ER. We hypothesize that QFL-specific CD8+ T cells and the mechanisms for generating the QFL-ligand are conserved sensors for detecting ERAAP-inhibition. Furthermore, disruption of these mechanisms in ERAAP-deficient mice may result in autoimmune obesity. We will accomplish these goals by (a) characterizing the ?? TCRs and the thymic developmental pathway for QFL-T cells to understand how these cells arise and acquire their unique functions, (b) defining the molecular steps for generating the QFL-ligand by analyzing the processing of Fam49a/b precursors into the FL9 peptide for loading the Qa-1 MHC Ib, and (c) defining the anatomical location(s) and function(s) of QFL-T cells in normal WT mice as well as their obese ERAAP-/- counterparts. Relevance Autoimmunity results when immune tolerance to self-antigens is disrupted. Understanding how self-antigens are produced by proteolytic enzymes will likely reveal potential ways to intervene in autoimmune disorders.